a) Field of the Invention
The invention is directed to a laser scanning microscope for providing three-dimensional and, more particularly, a process for controlling a laser scanning microscope with a tiltable fine focusing stage.
b) Description of the Related Art
A compact microscope stage for fine focusing is known from DE 19650392.
Further, a fast and highly precise fine focusing of an object in the direction of the optical axis z is made possible by a fine focusing stage comprising a frame, wherein a tongue which is connected with the frame on one side via a solid hinge or joint and is adjusted in the desired z-position on the other side with a high-precision galvanometer scanner moves in the frame.
The angular movement of the scanner is transmitted to the stage tongue via pulling means which run along the shaft of the scanner. The stage tongue and consequently the object held at the latter are accordingly tilted or rotated about the axis of the solid joint in the course of the fine focusing movement and are therefore adjusted at an angle xe2x89xa090xc2x0 to the optical axis. As a result of this tilting movement, a lateral offsetxcex94r of the object occurs in the xy-plane, this lateral offset having a significant magnitude particularly when, as is usually the case in practice, the object is not located exactly in the plane in which the axis of rotation of the stage tongue is situated. The axis of rotation A of the stage tongue is always oriented vertical to the optical axis as is shown in the figures
Formerly, a cable was always used as pulling means for transmitting the scanner movement to the stage tongue (DE 19650392). This involves numerous disadvantages: The cable is generally formed of several twisted wires because a single wire is either not flexible enough (too thick) or has insufficient loading capacity (too thin). When the cable which is tensioned by the force of gravity of the tongue and by the spring force of the spring joints runs along the scanning axis, these individual wires are alternately untwisted and then twisted together again. This causes a change in the length of the cable which transfers directly to the stage tongue. The precision and reproducibility with which the stage tongue can be adjusted in a desired position is accordingly lastingly impaired. Moreover, significant reverse backlash occurs which further impairs precision. The loading capacity of a cable of this kind is likewise very limited. Therefore, a stage of the type mentioned above is actually unsuitable in practice for fine focusing in the sub-100 nm range for which it was in fact designed.
A three-dimensional (3D) image which is recorded with a confocal laser scanning microscope (CLSM) is generally formed of a quantity (stack) of xy-sectional planes which are scanned in a pointwise manner and are drawn in successively in positions along the z-axis which succeed one another in a stepwise manner. The xy-raster of the individual sectional planes is generated in that a laser beam is deflected on the individual image points by means of a beam deflection system (e.g., by means of two galvanometer scanners). This raster is the same for all planes of the stack. The z-raster is generated in that the object is displaced in a parallel manner along the z-axis.
The computer-assisted image processing system of the CLSM arranges the image points belonging to the individual sectional planes one above the other in parallel manner, i.e., in a cuboid raster, in order to reconstruct the 3D image therefrom. The cuboid raster is shown in FIG. 1a. In this respect, it is required that all actual sectional planes in the object (1) extend exactly parallel to one another and (2) are arranged one exactly above the other along the z-axis. If these prerequisites are met, the image reconstructed by the computer is a real 1:1 mapping. However, this is only the case when the z-movement of the object is carried out along the optical axis.
On the other hand, if a z-stage with a galvanometer-controlled tilting tongue is used in the recording of the 3D image, these prerequisites are no longer met. Because of the tilting of the tongue, (1) the individual sectional planes are not parallel to one another and, due to the lateral offset which is a function of z, (2) they do not lie one above the other. The z-movement of the object with respect to the optical axis is carried out on a circular segment (FIG. 1b). The raster in the object (image field) is accordingly not a cuboid raster. In order to examine the deviations from the cuboid raster, it is sufficient to consider a plane which is oriented parallel to the optical axis. A plane E1 of this kind from the object raster and a plane E2 from the cuboid raster were placed one on top of the other in FIG. 2 in order to make the deviations visible.
If the 3D image of the scanned object is reconstructed by the computer in the conventional manner of the CLSM, the correlation of the raster points from the object of plane E1 to the cuboid raster of plane E2 is carried out in accordance with an algorithm shown by the arrows in FIG. 2. However, the 1:1 mapping is therefore no longer given. An unadvantageous distortion of the real image takes place through the transformation of the raster.
The primary object of the invention is the provision of a highly precise and distortion-free three-dimensional scanning by means of a laser scanning microscope.
In accordance with the invention, a fine focusing stage for a laser scanning microscope comprises a motor-adjustable or galvanometer-adjustable tilting tongue, wherein the tilting tongue is articulated at an adjusting portion by at least one flexible tape which is sturdy with respect to tension and is made from glass fiber-reinforced plastic.
Further in accordance with the invention, a process for displaying three-dimensional point distributions in a laser scanning microscope with a tiltable fine focusing stage comprising the steps of comparing an actual scanned first raster point distribution with a computer-generated second raster point distribution, utilizing the first raster points for display in the second raster point distribution when the raster points in both distributions correspond with one another, and forming intermediate values from points of the first raster point distribution lying in the vicinity of second raster points when there is no correspondence, wherein the position of these intermediate values corresponds to the position of the second raster points.
The use of a flexible tape in accordance with the invention obviates the disadvantages of cable discussed above.